The present invention relates to the field of molecular biology and immunology. More particularly, the invention relates to human thyrotropin receptor compositions. The invention is also related to diagnostic and therapeutic applications using human thyrotropin receptor compositions according to the present invention.
The most important of the diseases that cause thyrotoxicosis is Graves"" disease, also known as Parry""s or Basdow""s disease. However, not all hyperthyroidism is a result of Grave""s disease. Additionally, not all thyrotoxicosis is due to hyperthyroidism. Thyrotoxicosis is the clinical, biochemical and physiological result of sustained delivery of excessive quantities of thyroid hormones to the peripheral tissues. Hyperthyroidism is used to denote the situation where excess thyroid hormones stems from sustained thyroid hyperfunction.
There are a number of causes for thyrotoxicosis, the major ones include: Graves"" disease, toxic adenoma, toxic multinodular goiter, thyrotoxicosis factitia, ectopic thyroid, and thyroiditis. A portion of these are hyperthyroid based whereas the remaining portion is nonhyperthyroid. Graves"" disease is by far the most common cause of thyrotoxicosis, as well as the only autoimmune cause, it is therefore of extreme importance that an accurate assay be available to allow the differentiation between the auto immune and non-autoimmune varieties since the treatments, and pathogenesis differ so drastically. Bardin, Current Therapy in Endocrinology and Metabolism, B. C. Decker Inc. Toronto (1988).
Graves"" disease is a relatively common disorder that occurs at any age, but most often in the third and fourth decades. The disease is more frequent in women and the ratio of predominance in women may be as high as 7:1. The manifestations of Graves"" disease include one or more of the following: hyperthyroidism with diffuse goiter, ophthalmopathy, and dermopathy.
Thyrotropin, also known as thyroid stimulating hormone (TSH), is the primary hormone that regulates thyroid cell differentiated function and proliferation (Dumont et al., Adv. Cyclic Nucleotide Res. 14:479-489 (1981)). These effects are mediated by its interaction with the thyrotropin (TSH) receptor on the plasma membrane of thyroid cells. Thyroid stimulating auto-antibodies, the cause of thyrotoxicosis in Graves"" disease, mimic the actions of TSH by their interaction with the TSH receptor (Rees Smith et al., Endocr. Rev. 9:106-121 (1988)). In Graves"" disease, anti-TSH receptor auto-antibodies bind to the TSH receptor on the thyroid cell surface. Such binding causes unregulated stimulation of the thyroid cell which then produces excessive amounts of thyroid hormone.
The pathophysiological importance of the TSH receptor has motivated efforts directed to its characterization at both the protein as well as the genetic level. Hence, it has been found that it is a member of the G-protein-coupled receptor superfamily with glycoprotein hormone ligands, and that it is characterized by large, heavily glycosilated ectodomains with leucine-rich repeats encoded by several exons. (Gross et al., Biochem. Biophys. Res. Comm. 177:679-687 (1992); Tsai-Morris et al. J. Biol. Chem. 266:11355-11359 (1991); Koo et al., Endocrinol. 128:2297-2308 (1991)). Furthermore, characterization of cell bound TSH has identified two distinct functional forms. In one form, TSH is a single peptide receptor. In a second form, TSH is a heterodimer protein complex comprising subunit peptides A and B linked by disulfide bonds (Furmaniak et al., FEBS Letters 215:316-322 (1987), and Russo et al., Mol. Endocrinology 5:1607-1612 (1991)). Immunoprecipitation or immunoblotting analysis of mammalian cell extracts have shown variable proportions of single and two subunits forms, albeit the pathological significance of this finding is yet to be understood (Lossfelt et al., Proc. Natl. Acad. Sci. USA 895:3765-3769 (1992); Hata et al., Biochem. Biophys. Res. Comm. 164:1268-1273 (1989); Potter et al., BBRC 205:361-367 (1994); Chazenbalk et al., Endocrinology 137:4586-4591 (1996); Graves et al., Endocrinology 137:3915-3920 (1996)). Recent studies have suggested that subunits A and B are the product of intramolecular cleavage at two separate sites, with the concomitant loss of a TSH fragment, referred to in the literature as peptide C (expected to correspond to an approximately 5 KDa plypeptide fragment) (Chazenbalk, et al., Endocrinology 138:2893-2899 (1997)). To date however, the sites of cleavage, the kinetics of the cleavage as well as the enzyme(s) involved have yet to be elucidated. In addition, the physiological and/or pathological implications of peptide C, if any, have yet to be investigated.
The difficulties encountered in characterizing Graves"" disease at the molecular level parallel the obstacles encountered in devising sensitive and specific diagnostic assays. Thyroid stimulating auto-antibodies, originally named xe2x80x9clong-acting thyroid stimulatorsxe2x80x9d or LATS, were measured with a bioassay system (McKensie) using mouse thyroid tissue. This assay was considered to be fairly specific for Graves"" disease, although positive results were found in a few patients with Hashimoto""s disease and occasionally, albeit in smaller titers, in other unrelated conditions. In addition, this assay could only detect 40-45% of patients with Graves"" disease. Notably, there was no correlation between titer and the severity of the disease.
More recently, other related IgG antibodies were found that prevent adsorption of LATS onto human thyroid tissue and as a group were therefore called xe2x80x9cLATS protector.xe2x80x9d This abnormal IgG antibody (or antibodies) could be demonstrated by three different assay techniques. One technique used high-titer LATS antibody as the indicator system whose endpoint was to show that antibody in patient serum blocked uptake of the LATS antibody by human thyroid tissue. A second technique, also known as xe2x80x9cTSH-binding inhibiting immunoglobulinxe2x80x9d or TBII assay, measured, the ability of patient antibody to inhibit or block the binding of radiolabeled TSH to human or animal thyroid acinar cell membranes. A third technique measured the ability of the antibody to stimulate human or animal thyroid acinar cell membrane-bound adenylate cyclase, producing increased cyclic adenosine monophosphate (AMP) activity. The original assay method used human thyroid tissue, so the antibody was originally called xe2x80x9chuman thyroid-stimulating immunoglobulin.xe2x80x9d Since animal thyroid tissue can be used, the assay now is simply called xe2x80x9cthyroid-stimulating immunoglobulinxe2x80x9d (TSI). For example, one modification of the TSI uses special TSH-dependent FRTL-5 rat tissue culture thyroid cells. The LATS-protector assay has been reported to detect that about 75%-80% (range , 60%-90%) of patients with Graves"" disease. The LATS-protector assay is very complicated. The TBII technique is reported to detect only about 70%-80% (range, 39%-100%) of Graves"" disease, and the TBI detects only about 75%-80% (range, 18%-100%). Neither the TBII nor the TBI are simple or easy.
In addition to the low sensitivity inherent to the systems used, most laboratories using either technique use xe2x80x9chomemadexe2x80x9d reagents, which accounts for much of the great variation in sensitivity reported in the literature. Furthermore, since many clinical laboratories base their test performance claims on data from one or more research laboratories using the same technique but separate reagents additional levels of variability are compounded upon standardization. Besides sensitivity, it is necessary to question specificity, since different laboratories may find different numbers of false positive results when patients with hyperfunctioning thyroid nodules, nonfunctioning nodules or goiter, thyroiditis, autoimmune disorders, and clincially normal status are tested.
At present, the TSI assay is used mainly for patients with borderline or conflicting evidence of Graves"" disease, patients who have some condition that affects the results of other tests, or patients who have xe2x80x9cisolated Graves"" ophthalmopathyxe2x80x9d (a condition in which all standard thyroid tests are normal). However, a negative test result in most laboratories does not completely exclude the diagnosis, and there is still remain a possibility of false positive results.
One of the obstacles to the development of more sensitive and specific diagnostic assays is represented by the reported instability of isolated TSH receptor preparations necessary for the assays. Instability is most likely attributable to the harsh conditions inherent to the purification process during which the A and B subunit polypeptide backbones are likely to disassociate. Thus, there remains a need for specific and sensitive assay systems.
At present, there is no therapy that can cure Grave""s disease. There are several current treatments with many drawbacks. In one treatment for Graves"" disease, drugs are administered that block thyroid hormone synthesis. These drugs are administered for many months or years, while waiting for a spontaneous emission of the thyroid overactivity. Another radical treatment requires ablation of part or all of the thyroid by surgery or radioactive iodine. This commonly leads to hypothyroidism and the need for life-long administration of thyroid hormone.
While there are some treatments available, none is available which is directed primarily at the underlying immunologic cause of Graves"" disease. Hence, there is a need for the development of immunological approaches to the treatment and further elucidation of Graves"" disease.
The invention provides novel methods and compositions for the diagnosis and treatment of Graves"" disease. The present inventors have localized the two sites at which, by intramolecular cleavage, the TSH receptor A and the B subunits are formed. Thus, the invention provides compositions and methods for a convenient and economical source of recombinant TSH receptor. The invention also provides enriched TSH receptor compositions and methods of using the same. Finally, the invention provides methods for using such compositions as analytical and diagnostic tools, as potentiators of transgenic animal studies and for gene therapy approaches, and as potential therapeutic agents.
With seven transmembrane domains, the TSH receptor belongs to a family of G protein-coupled receptors, including the receptors for LH CG, substance K, rhodopsin, serotonin, as well as the xcex12-, xcex21- and xcex22-adrenergic and muscarinic cholinergic receptors (McFarland et al., Science 245:494-499 (1989); Loosfelt et al., Science 245:525-528 (1989)). In contrast to the short extracellular domains recognized by the smaller ligands such as adrenergic and cholinergic agents, the extracellular domain of the TSH receptor is much larger and more complex. This finding is consistent with the complexity of the glycoprotein hormones which are approximately 30 kDa in size, and suggests that the extracellular domain plays an important role in hormone binding and signal transduction.
The identification of the cleavage sites in the TSH receptor now opens the way for future studies to answer many questions that have remained unanswered for a number of years. These questions include (a) the identification of the site(s) of binding (epitopes) of stimulatory and inhibitory anti-TSH receptor antibodies present in the sera of patients with autoimmune thyroid disease, and the relationship between these binding sites and that for TSH, (b) the determination of the mechanism of signal transaction by which TSH increases adenylate cyclase activity in thyroid cells; and (c) the mechanism by which continued TSH stimulation leads to a decrease in TSH receptor-coupling with Gs and reduced adenylated cyclase activation (homologous desensitization).
The production of enriched recombinant TSH receptor preparations also makes possible new treatments for thyrotoxicosis which would not have the drawbacks of current therapies.
In a first aspect, the invention provides recombinant nucleic acid sequences in a replicatable vector encoding for the thyrotropin receptor, or a functional or chemical derivative thereof, having a deletion capable of inhibiting cleavage at site 1. In a preferred embodiment, the replicatable vector is an expression vector.
In a second aspect, the instant invention provides a recombinant thyrotropin receptor protein, or a functional or chemical derivative thereof, including a deletion capable of inhibiting cleavage at site 1. In an embodiment of the invention the deletion encompasses amino acids 317 to 366.
In a third aspect, the invention provides a recombinant nucleic acid sequence in a replicatable vector encoding for the thyrotropin receptor, or a functional or chemical derivative thereof, including a sequence encoding a cleavage-resistant moiety capable of resisting cleavage at cleavage site 2. In a preferred embodiment of this invention, the cleavage-resistant moiety is a consensus sequence for an N-linked glycosylation site.
In another embodiment of the invention, the replicatable vector is an expression vector. In yet another embodiment, the invention also provides a host cell comprising recominant DNA molecules according to the invention.
In a fourth aspect, the instant invention provides a recombinant thyrotropin receptor protein, or a functional or chemical derivative thereof, including a sequence encoding a cleavage-resistant moiety capable of resisting cleavage at cleavage site 2 in a replicatable vector.
In a preferred embodiment according to this aspect of the invention, the cleavage-resistant moiety spans residues 367 to 369. In an embodiment of the invention, the recombinant nucleic acid sequence includes both a deletion capable of inhibiting cleavage at site 1, and a sequence encoding a cleavage-resistant moiety capable of resisting cleavage at cleavage site 2 in a replicatable vector. In a preferred embodiment of the invention, the recombinant nucleic acid sequence including both a deletion capable of inhibiting cleavage at site 1, and a sequence encoding a cleavage-resistant moiety capable of resisting cleavage at cleavage site 2 is an expression vector. In yet another embodiment of the invention, the recombinant nucleic acid sequence including both a deletion capable of inhibiting cleavage at site 1, and a sequence encoding a cleavage-resistant moiety capable of resisting cleavage at cleavage site 2 is in a host cell. In another embodiment, the invention also provides a recombinant thyrotropin receptor protein including both a deletion capable of inhibiting cleavage at site 1, and a sequence encoding a cleavage-resistant moiety capable of resisting cleavage at cleavage site 2. In a most preferred embodiment, the invention provides a recombinant thyrotropin receptor protein including both a deletion corresponding to amino acids 317 to 366, capable of inhibiting cleavage at site 1, and a cleavage-resistant moiety between amino acid residues 367 to 369 capable of resisting cleavage at cleavage site 2. In another most preferred embodiment, the invention provides a recombinant thyrotropin receptor protein including both a deletion corresponding to amino acids 317 to 366, capable of inhibiting cleavage at site 1, and a consensus sequence for an N-linked glycosylation site, capable of resisting cleavage at cleavage site 2.
In a fifth aspect, the invention provides a method of producing thyrotropin receptor by culturing the transformed cell of the present invention under conditions allowing expression of thyrotropin receptor, and recovering said thyrotrophin receptor.
In a sixth aspect, the present invention provides for an antibody against the thyrotropin receptor of the invention. In an embodiment, the invention provides a method of detecting thyrotropin receptor in a sample by contacting the sample with a labeled antibody crossreactive with the thytropin receptor, so as to form a detectable complex between the thyrotropin receptor sample and the antibody. In an additional embodiment, there is provided a kit for the detection of thyrotropin receptor in a sample, including container means with one or more containers, wherein one of said containers comprises detectably labeled antibody against thyrotropin receptor.
Moreover, a method of detecting antibodies to thyrotropin receptor in a sample is provided according to the present invention, including contacting a sample with detectably labeled recombinant thyrotropin receptor, so as to form a complex between the TSH receptor antibodies in the sample and the detectably labeled recombinant TSH receptor, and detecting the complexed antibody. In an additional embodiment, there is provided a kit for the detection of antibodies to thyrotropin receptor in a sample, including container means with one or more containers. According to the embodiments, one of the containers includes detectably labeled TSH receptor.
An additional aspect of the current invention is a method for differentiating between auto immune and non-auto immune varieties of thyrotoxicosis comprising an assay specific for either thyrotropin receptor, anti-thyrotropin receptor autoantibodies or functional or chemical derivatives thereof.
Another embodiment of the current invention is a method of treating thyrotoxicosis with pharmacologically effective amounts of recombinant thyrotropin receptor, or a functional or chemical derivative thereof.
In an additional embodiment, a thymus-derived lymphocyte (T cell) is provided which is specific for the autoimmune T-cell receptor (TCR) for TSH receptor. Another embodiment of the invention provides for a pharmaceutical preparation comprising a T cell which is specific for the autoimmune TCR for TSH receptor. In yet another embodiment of the current invention, a method of treating autoimmune thyrotoxicosis is provided comprising the use of a pharmaceutical preparation comprising a T cell which is specific for the autoimmune TCR for TSH receptor.
Another embodiment provides a peptide which is specific for the autoimmune TCR for TSH receptor. Another embodiment of the invention provides for a pharmaceutical preparation comprising a peptide which is specific for the autoimmune TCR for TSH receptor. In yet another embodiment of the current invention, a method of treating autoimmune thyrotoxicosis is provided including the use of a pharmaceutical preparation comprising a peptide which is specific for the autoimmune TCR for TSH receptor.
In an additional embodiment, there is provided suppressor T cells specific for anti-thyrotropin receptor auto-antibodies. Another embodiment provides a pharmaceutical preparation including suppressor T cells specific for anti-thyrotropin receptor auto-antibodies. Yet another embodiment provides a method of treating thyrotoxicosis with a pharmaceutical preparation comprising T cells specific for anti-thyrotropin receptor auto-antibodies.
These and other non-limiting embodiments of the present invention will be apparent to those of skill from the following detailed description of the invention.